Hrq1/RECQL4 regulation is critical for preventing aberrant recombination during DNA intrastrand crosslink repair and is upregulated in breast cancer

Human RECQL4 is a member of the RecQ family of DNA helicases and functions during DNA replication and repair. RECQL4 mutations are associated with developmental defects and cancer. Although RECQL4 mutations lead to disease, RECQL4 overexpression is also observed in cancer, including breast and prostate. Thus, tight regulation of RECQL4 protein levels is crucial for genome stability. Because mammalian RECQL4 is essential, how cells regulate RECQL4 protein levels is largely unknown. Utilizing budding yeast, we investigated the RECQL4 homolog, HRQ1, during DNA crosslink repair. We find that Hrq1 functions in the error-free template switching pathway to mediate DNA intrastrand crosslink repair. Although Hrq1 mediates repair of cisplatin-induced lesions, it is paradoxically degraded by the proteasome following cisplatin treatment. By identifying the targeted lysine residues, we show that preventing Hrq1 degradation results in increased recombination and mutagenesis. Like yeast, human RECQL4 is similarly degraded upon exposure to crosslinking agents. Furthermore, over-expression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. Using bioinformatic analysis, we observe that RECQL4 overexpression correlates with increased recombination and mutations. Overall, our study uncovers a role for Hrq1/RECQL4 in DNA intrastrand crosslink repair and provides further insight how misregulation of RECQL4 can promote genomic instability, a cancer hallmark.

We agree with the reviewer that there were differences in the ubiquitin partner depending upon whether ubiquitin itself or Hrq1 was immunoprecipitated. An important difference between these experiments is that the Hrq1 pull down used 25% of the yeast culture compared to the ubiquitin pull down. Therefore, one possibility is that more Hrq1 species can be visualized. Furthermore, the ubiquitin pulldown relies on massive over-expression of ubiquitin and perhaps that creates differences compared to analyzing the endogenous Hrq1 protein that is not over-expressed. We have made a note of this in the Results. Fig. 2a Hrq1 appeared to be already unstable in Rad16 deleted cells even in the absence of cisplatin. Why? Again, I do not see much difference in Hrq1 stability of wt and rad16 deleted cells in these images, while graphs shows differently.

In
We agree with the reviewer that we should explain these results more clearly. Hrq1 is both cell cycle regulated and regulated upon exposure to intrastrand adducts. Therefore, there may be multiple independent modes of regulation. We find that deleting RAD16 stabilized Hrq1 protein levels upon cisplatin exposure specifically (Figure 2). There could be other ubiquitin ligases that regulate Hrq1 protein levels that are DNA damage independent. We have clarified this point in the Results. Fig. 3a Alpha factor arrested cells are not real G1 cells. Alpha factor arrests cell cycle before start of cell cycle at G1, and expression of many genes involved in cell proliferation can be reduced in alpha factor arrested cells. You can analyze the G1 cells in the next cell cycle or you can use other methods for cell synchronization such as nocodazole arrest and release to make sure that changes in the level of proteins indeed occur in G1.

In
We respectfully disagree with the reviewer as alpha factor is extensively used to arrest cells in G1. Regardless, our main conclusion is to demonstrate that Hrq1 expression peaks before Clb2 expression.

In Fig. 3f
Differences in cisplatin sensitivity between rev1 deletion and hrq1 and rev1 deletion are so marginal, and only found in a single plate (20 ug/ml cisplatin). I am very skeptical to make any conclusion based on these observations. We agree with the reviewer that the synthetic sickness observed between hrq1∆ rev1∆ double mutant is subtle but highly reproducible. We provide additional evidence that siRNA knockdown of human RECQL4 also exhibits a subtle, but highly reproducible, growth defect upon siRNA knockdown of REV1. These results are consistent with Groocock et al 2012, who showed that fission yeast hrq1∆ cells exhibit more spontaneous mutations. Together, these data support our observations here (Figure 5c, 5d) and we now include these findings from Groocock in the paper. Fig. 3 and Sup. Fig. 3, yeast cells are much more sensitive to cisplatin than MMC at the same concentration. Therefore, the result obtained by using the same concentration of MMC is not conclusive.

of yeast cells to cisplatin and MMC in
Unfortunately, we cannot directly compare doses of different damaging agents as they cause different types of DNA damage and their effects not necessarily the same on hrq1∆ cells as observed by the different sensitivity. However, we have provided multiple cisplatin and MMC doses for analysis here (Supplemental Figure 3a). Figure 4c The levels of Hrq1 in WCE are different. If you want to directly compare the intensities of ubiquitinated proteins in IP materials, you have to use the same amount of extracts containing the same amount of target proteins.

In
We agree with the reviewer that the levels of Hrq1 in WCE are different either due to pipetting error or to degradation from cisplatin exposure. To account for the difference in the WCE, we normalized the IP to the WCE, which mitigates any of these technical differences.

In Figure 5c
You need to show the cisplatin sensitivity of double knock-down of RecQL4 and any one functioning in error-free PRR pathway to suggest the role of RecQL4 in the error-free PRR.
We agree with the reviewer that double knock-down of RecQL4 and a gene functioning in the error-free PRR pathway would be very informative. We attempted to knockdown both HLTF and SHPRH, the homologs of Rad5. However, the results were inconclusive. Knockdown of HLTF alone leads to decreased cellular proliferation even in the absence of damage, which confounded our results when treated with cisplatin. On the other hand, knockdown of SHPRH seems to result in cisplatin resistance (Reviewer Figure 1). More extensive analysis is needed to elucidate this complex pathway and is beyond the scope of the current paper. Therefore, we have softened the language discussing RecQL4 function with error-free PRR in the Results.
Reviewer Figure 1 Legend: U2OS cells were transfected with either siCon, siRECQL4, siHLTF, and/or siSHPRH for 48 hours and 300 cells per well were plated in triplicate. The following day the medium was replaced with fresh medium with or without the indicated dose of cisplatin. After 24 hours, the medium was removed, the cells were washed, and fresh medium was added. The cells grew for 17 days before crystal violet staining. The experiment was repeated in triplicate with standard error plotted and the colonies were normalized to the untreated siCon. Fig. 6. Rad51 staining is poor, and cisplatin treatment did not increase Rad51 foci in immunostaining and tail moments in comet assay even in U2OS cells. Therefore, I do not think that these assays are reliable in this condition.

In
We agree with the reviewer that tail moments in the comet assay did not exhibit more DSBs. Since we were focusing on bypass of replicative damage and not direct DSB repair, we did not analyze the cells at a time point where DSBs would form. Also, treatment with S1, which should result in increased DSBs, lead to increased tail moments, which indicates the assay worked as expected. We have clarified this point in the Results.
With regards to the RAD51 staining, we have now included improved image quality so that the foci are more easily visualized (New Figure 6a). The RAD51 foci are analyzed automatically in a blinded fashion, and this enables appropriate thresholding and background to be uniformly assessed. We have expanded upon this analysis in the Materials & Methods.

New Figure 6a Legend:
Overexpression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. U2OS cells were transfected with an empty plasmid or a plasmid expressing RECQL4 or RECQL4-K508A under a CMV promoter. The cells were either mock or cisplatin treated for one hour and after a two-hour recovery, imaged for RAD51 foci or DAPI by immunofluorescence. RAD51 foci was quantified from 200 cells per condition for each experiment. The experiment was performed three to five times and the median was graphed. Representative images are shown.

Reviewer 2
Major Comments: 1) Figure 3A: Whereas it is obvious from the Western blot images provided that Clb2 peaks at 60 minutes, the same is not true for the protein of interest, Hrq1-9-Myc. Instead, Hrq1-9-Myc appears to plateau at 40 minutes. To strengthen the conclusion that Hrq1 protein levels peak at 40 minutes, I recommend quantification of the results in these blots as in Figures 1C and 2A.
We have now quantitated the western blots from Figure 3a and indeed Hrq1 begins to peak prior to Clb2 (New Figure 3a). Quantification from three separate experiment, the mean with SEM is shown. The cell cycle stage was analyzed FACS.
Between 0 and 40 minutes, the data for Hrq1-9-Myc suggest that Hrq1 is upregulated and then plateaus. For Hrq1 levels to drop again during the next cell cycle, one would expect degradation. It would be helpful to see this Western blot data extended to additional time points into the second cell cycle.
We agree with the reviewer that it would be ideal to analyze a second cell cycle. However, yeast cells lose their synchronization over time and therefore, we can only reliably analyze cells for one cell cycle. Figure 3A leads one to believe that these experiments have not been repeated. Thus, to make this claim that Hrq1 levels peak during S/G2, I believe that the data needs to quantified, repeated, and extended to later time points, such that the return to G1 levels is clear. Figure 3a, were repeated a minimum of three times (exception is the DRR assay where we had a technical issue with one of the replicates). We have now made that clear in the Figure 3a legend and quantified the data in the western blots from multiple experiments. Figure 4D: We agree with the reviewer and have included a schematic of the recombination reporter assay used in Figure 4d.

Based on the diagram in Godin et al., it is clear that Leu+ colonies in this system can result from repair either through homologous recombination or single-strand annealing. These two possibilities can be distinguished by determining whether the resulting colonies are Ura+ or Ura-. The implications of these two outcomes for this study should be presented in the text. Moreover, the results of this analysis showing whether damage is repaired primarily through recombination or single-strand annealing should be included in the figure.
We have now included the rates of gene conversion and single-strand annealing observed upon over-expression of HRQ1 or HRQ1-7KR. We find that both gene conversion and single-strand annealing are elevated upon cisplatin exposure (New Figure 4e). represents p<0.01, *** represents p<0.001 e) The average rates of GC and SSA for each condition are shown, average rates with SEM are graphed.

3) Figures 5C and D: Figures 5C and D:
The manuscript does not show that the siRNA is specific to RECQL4 or REV1 by back complementation with siRNA resistant plasmids encoding RECQL4 or REV1. This is a relatively standard control for these types of experiments, and would create more confidence in the results, as it would show that the results are specific to RECQL4 and/or REV1.
We agree with the reviewer that using siRNA resistant plasmids to complement our assays are an important control. We tried to create a siRNA resistant plasmid for REV1, but we had difficulty cloning it. We also tried to buy an existing plasmid (https://www.addgene.org/153001/) to subclone into our vector but when we checked the gene sequence, the REV1 sequence was significantly truncated in the original plasmid. Unfortunately, due to the timing, we were not able to perform the REV1 complementation experiments. As for RECQL4, the siRNA sequence we used was from the Vilhelm Bohr's laboratory and has been used in multiple publications: We thank the reviewer for this suggestion as we were unaware of the study by Löbrich and colleagues. We have now included discussion that use of SDSA as a predominant repair mechanism is not universal and cell line dependent in the Discussion. Figure 6A: Quantifying only the total number of RAD51 foci present at a single time point is not an appropriate method to determine whether there is hyper-recombination in a given mutant. There are many alternative explanations that could explain the results observed, such as increased DNA damage, or RAD51 persistence due to unresolved recombination intermediates or lack of repair. The conclusions in this section should be revised to reflect this.

5)
We agree with the reviewer and have revised the conclusions of this section to include these other possibilities as to why more RAD51 foci may be observed.
The authors should also indicate in their quantitation whether the difference in RAD51 foci between the empty vector and the RECQL4-KA mutant is statistically significant.
We have now included statistical analysis RAD51 foci compared with the empty vector and the RECQL4-KA mutant, and it is indeed statistically significant by t-test (p < 0.01). Figure 7E: It has been reported that the majority of tumors overexpress RAD51 (e.g. Klein 2008 DNA Repair). Therefore, the implications of this finding are somewhat overstated, and should be revised.

6)
We agree with the review and have now softened this statement. We have now included discussion of the Lu et al 2017 Nature Comms paper (p. 21). The primary difference between the Lu paper and our study is that they looked at RECQL4 roles during DSB repair, while our studies examined RECQL4 role during bypass of fork stalling lesions. Corrected and sample size is now acknowledged. Corrected.

4) Discussion:
The manuscript states, "Furthermore, we find that Hrq1 is regulated by the UPS during instrastrand crosslink repair and that misregulation of Hrq1 protein levels results in increased recombination and mutagenesis. We extend this analysis to mammalian cells and show that like yeast, human RECQL4 protein levels are depleted in response to DNA crosslinks… Together our studies demonstrate a conserved role for the RECQL4 protein family in crosslink repair whose levels must be fine-tuned to prevent excess recombination and mutations." See comments on Figures 4 and 6 above. I believe the data